Laminin mediated alveolar cell mechano-transduction

层粘连蛋白介导的肺泡细胞机械转导

基本信息

批准号：
7435396
负责人：
JONATHAN C. JONES
金额：
$ 32.88万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7435396
关键词：
ATP phosphohydrolase Acute Adenoviruses Adult Respiratory Distress Syndrome Alveolar Alveolar Cell Animals Antibodies Apoptosis Basement membrane Binding Biological Assay CD29 Antigen Cell Adhesion Cell physiology Cell surface Cell-Matrix Junction Chemicals Complex Cultured Cells Data Edema Electroporation Epithelial Epithelial Cells Excision Exposure to Extracellular Matrix Generations Genes Genetic Transcription Helper Viruses Hour In Vitro Inflammatory Integrins K ATPase Knock-out Laminin Lung Measures Mechanical ventilation Mechanics Mediating Membrane Proteins Mitogen-Activated Protein Kinases Mitogens Molecular Mus Na(+)-K(+)-Exchanging ATPase Newborn Respiratory Distress Syndrome Oxidants Patients Plasmids Play Protein Isoforms Rattus Respiratory Failure Respiratory physiology Role Stream Stretching Technology Testing Tidal Volume Transgenic Organisms Treatment Protocols Wound Healing in vivo inhibiting antibody inhibitor/antagonist insight laminin alpha 3 lung injury mature animal migration mortality promoter receptor recombinase research study

项目摘要

Mechanical ventilation worsens pre-existing lung injury in animals and contributes to mortality in patients with the acute respiratory distress syndrome (ARDS). Mechanical stretch of cultured ceils increases oxidant generation, induces the expression of pro-inflammatory genes, inhibits epithelial wound healing and induces apoptosis. The mechanism(s) by which alveolar epithelial cells sense cyclic stretch, however, have not been elucidated. Preliminary experiments suggest that basement membrane proteins may play an important role in mediating mechanosignal transduction in the lung. In particular, cultured alveolar ceils secrete a matrix rich in laminin containing an alpha3 subunit The alpha3 subunit-containing laminin organizes into a fibrillar arrays. Moreover, a transient increase in mitogen activated protein kinase (MAPK) in rat alveolar cells induced by stretch is inhibited by an antibody against the alpha3 laminin subunit globular domain and by antibodies that perturb the function of beta1 integrin. These data provide support for a hypothesis that mechanosignaling in alveolar cells is mediated by a laminin/integrin complex. We will test this hypotheis in three specific aims. In aim 1, we will determine the functional consequences of alveolar epithelial cell adhesion to laminins in the extracellular matrix. Specifically, we propose to determine laminin isoforms expressed by alveolar cells in vitro and in vivo. In addition, we will assess the role of integrin receptors in alveolar attachment, spreading and migration on different laminin isoforms. In aim 2, we will determine whether a laminin/integrin interaction is responsible for mechanosignal transduction in cultured cells. We will examine the effects of inhibitors of the laminin/integrin interaction in primary cultures of alveolar epithelial cells exposed to cyclic stretch focusing on MAP kinase activation and its down stream consequences on the activity and expression of the Na+-K+-ATPase. In aim 3 we will determine whether the alpha3 laminin subunit is responsible for mechanosignal transduction in vivo. We will develop a transgenic lung-specific conditional knockout of alpha3 laminin using Cre recombinase mediated excision. Lung function will be evaluated in adult animals lacking the alpha3 laminin. These studies will provide new insight into the role of extracellular matrix in regulating lung cell physiology.

机械通气会恶化动物的肺损伤，并导致急性呼吸遇险综合征（ARDS）患者的死亡率。培养的天花板的机械拉伸会增加氧化剂的产生，诱导促炎基因的表达，抑制上皮伤口愈合并诱导凋亡。但是，尚未阐明肺泡上皮细胞感应循环拉伸的机制。初步实验表明，基底膜蛋白可能在介导机械信号中起重要作用肺部转导。特别是，培养的肺泡天花板分泌一个富含层粘连蛋白的基质，其中含有alpha3亚基，含有alpha3亚基的层粘连蛋白会组织成纤维阵列。此外，针对Alpha3层粘连蛋白亚基球状结构域的抗体抑制了拉伸诱导的大鼠肺泡细胞中有丝分裂原活化蛋白激酶（MAPK）的瞬时增加，并且通过抗体抗体的抗体抗体。这些数据为假设提供了支持，即肺泡细胞中的机械信号是由层粘连蛋白/整合素络合物介导的。我们将以三个特定目标来测试这一假设。在AIM 1中，我们将确定肺泡上皮细胞粘附在细胞外基质中的功能后果。具体而言，我们建议确定肺泡细胞在体外和体内表达的层粘连蛋白同工型。此外，我们将评估整联蛋白受体在牙槽附着中的作用，在不同的层粘连蛋白同工型上扩散和迁移。在AIM 2中，我们将确定层粘连蛋白/整合素相互作用是否负责培养细胞的机械信号转导。我们将研究层粘连蛋白/整合素相互作用在暴露于循环拉伸的牙槽上皮细胞的原发性培养物中的抑制剂，集中在MAP激酶激活上及其对Na+-K+-ATPase的活性和表达的后果。在AIM 3中，我们将确定α3层粘连蛋白亚基是否负责体内机械信号转导。我们使用CRE重组酶介导的切除术，将开发α3层粘连蛋白的转基因肺特异性敲除。在缺乏α3层粘连蛋白的成年动物中，将评估肺功能。这些研究将为细胞外基质在调节肺部细胞生理学中的作用提供新的见解。